Peritoneal Microenvironment Promotes Appendiceal Adenocarcinoma Growth: A Multi-omics Approach Using Patient-Derived Xenografts.

Appendiceal adenocarcinoma (AA) is unique from other gastrointestinal malignancies in that it almost exclusively metastasizes to the peritoneal cavity. However, few studies have investigated the molecular interaction of the peritoneal microenvironment and AA. Here, we use a multi-omics approach with orthotopic and flank-implanted patient-derived xenografts (PDX) to study the effect of the peritoneal microenvironment on AA. AA tumors implanted in the peritoneal microenvironment tended to grow faster and displayed greater nuclear expression of Ki-67 relative to the same tumors implanted in the flank. Comparing the tumor-specific transcriptome (excluding stromal transcription), the peritoneal microenvironment relatively upregulated genes related to proliferation, including MKI67 and EXO1. Peritoneal tumors were also enriched for proliferative gene sets, including E2F and Myc Targets. Proteomic studies found a 2.5-fold increased ratio of active-to-inactive phosphoforms of the YAP oncoprotein in peritoneal tumors, indicating downregulation of Hippo signaling. IMPLICATIONS: The peritoneal microenvironment promotes growth of appendiceal tumors and expression of proliferative pathways in PDXs.

Nerve sheath tumor in the forearm of a common marmoset (Callithrix jacchus).

Tumors of the skin and subcutaneous tissues are uncommon in marmosets. In this report, we describe the gross, histopathology, and immunohistochemical findings of a nerve sheath tumor that arose in the left forearm of an adult female marmoset (Callthrix jacchus).

Neutrophil Elastase Remodels Mammary Tumors to Facilitate Lung Metastasis.

Metastatic disease remains the leading cause of death due to cancer, yet the mechanism(s) of metastasis and its timely detection remain to be elucidated. Neutrophil elastase (NE), a serine protease secreted by neutrophils, is a crucial mediator of chronic inflammation and tumor progression. In this study, we used the PyMT model (NE+/+ and NE-/-) of breast cancer to interrogate the tumor-intrinsic and -extrinsic mechanisms by which NE can promote metastasis. Our results showed that genetic ablation of NE significantly reduced lung metastasis and improved metastasis-free survival. RNA-sequencing analysis of primary tumors indicated differential regulation of tumor-intrinsic actin cytoskeleton signaling pathways by NE. These NE-regulated pathways are critical for cell-to-cell contact and motility and consistent with the delay in metastasis in NE-/- mice. To evaluate whether pharmacologic inhibition of NE inhibited pulmonary metastasis and phenotypically mimicked PyMT NE-/- mice, we utilized AZD9668, a clinically available and specific NE inhibitor. We found AZD9668 treated PyMT-NE+/+ mice showed significantly reduced lung metastases, improved recurrence-free, metastasis-free and overall survival, and their tumors showed similar molecular alterations as those observed in PyMT-NE-/- tumors. Finally, we identified a NE-specific signature that predicts recurrence and metastasis in patients with breast cancer. Collectively, our studies suggest that genetic ablation and pharmacologic inhibition of NE reduces metastasis and extends survival of mouse models of breast cancer, providing rationale to examine NE inhibitors as a treatment strategy for the clinical management of patients with metastatic breast cancer.

High-fat diet, but not duration of lactation, increases mammary gland lymphatic vessel function and subsequent growth of inflammatory breast cancer cells.

Inflammatory breast cancer (IBC) presents as rapid-onset swelling and breast skin changes caused by tumor emboli in the breast and breast skin lymphatics. IBC has been linked with obesity and duration of breastfeeding, but how these factors affect IBC tumor progression is not clear. We modeled the simultaneous effects of diet and weaning in mice on in vivo lymphatic function; on IBC tumor growth; and on aspects of the mammary gland microenvironment before and after IBC (SUM149) xenograft inoculation. We hypothesized that weaning status and diet would have synergistic effects on lymphatic function and the breast microenvironment to enhance IBC tumor growth. Changes in lymphatic structure and function were characterized with in vivo near-infrared fluorescence (NIRF) imaging. Mice were fed either a high-fat diet (HFD; 60 kcal%) or a normal/low-fat diet (LFD; 10 kcal%), bred twice, and subjected to either normal-duration nursing (NW) or forced weaning (FW). SUM149 IBC tumors were implanted at 14 months; images were obtained before and after implantation. Multiparous mice fed HFD showed increased pre-tumor lymphatic pulsing in both the FW and NW groups relative to mice fed LFD. HFD promoted tumor growth independent of weaning time (P = 0.04). Pre-tumor lymphatic pulsing was associated with tumor volume at 8 weeks (P = 0.02) and was significantly correlated with expression of the lymphatic tracking ligand CCL21 (P = 0.05, Table 1). HFD significantly increased the numbers of monocyte-derived IBA1+, CD163+, and CD11c+ cells (P < 0.0001, P < 0.0001, P = 0.0005) in the contralateral, non-tumor-bearing mammary gland. Numbers of lymphangiogenic podoplanin+/IBA1+ macrophages were increased in the ducts of HFD and FW mice (all P < 0.003). HFD in nulliparous mice had a similar increase in lymphatic pulsing at 14 weeks (P = 0.006), indicating that this functional change was independent of parity. We conclude that HFD induced increases in mammary gland lymphatic function, assessed as pulsing rate before tumor initiation, and correlated with inflammation in the mammary gland and increased SUM149 tumor growth. The relationship between diet, lymphatic pulsing, and tumor growth warrants further investigation.

Intraperitoneal Paclitaxel Is a Safe and Effective Therapeutic Strategy for Treating Mucinous Appendiceal Adenocarcinoma.

Appendiceal adenocarcinomas (AA) are a rare and heterogeneous mix of tumors for which few preclinical models exist. The rarity of AA has made performing prospective clinical trials difficult, which has partly contributed to AA remaining an orphan disease with no chemotherapeutic agents approved by the FDA for its treatment. AA has a unique biology in which it frequently forms diffuse peritoneal metastases but almost never spreads via a hematogenous route and rarely spreads to lymphatics. Given the localization of AA to the peritoneal space, intraperitoneal delivery of chemotherapy could be an effective treatment strategy. Here, we tested the efficacy of paclitaxel given by intraperitoneal administration using three orthotopic patient-derived xenograft (PDX) models of AA established in immunodeficient NSG mice. Weekly intraperitoneal paclitaxel treatment dramatically reduced AA tumor growth in all three PDX models. Comparing the safety and efficacy of intravenous with intraperitoneal administration, intraperitoneal delivery of paclitaxel was more effective, with reduced systemic side effects in mice. Given the established safety record of intraperitoneal paclitaxel in gastric and ovarian cancers, and lack of effective chemotherapeutics for AA, these data showing the activity of intraperitoneal paclitaxel in orthotopic PDX models of mucinous AA support the evaluation of intraperitoneal paclitaxel in a prospective clinical trial. SIGNIFICANCE: The activity and safety of intraperitoneal paclitaxel in orthotopic PDX models of mucinous appendiceal adenocarcinoma supports the evaluation of intraperitoneal paclitaxel in a prospective clinical trial of this rare tumor type.

Antitumor activity of intraperitoneal paclitaxel in orthotopic patient-derived xenograft models of mucinous appendiceal adenocarcinoma.

Appendiceal adenocarcinomas (AAs) are a rare and heterogeneous mix of tumors for which few preclinical models exist. The rarity of AA has made performing prospective clinical trials difficult, and in part because of this AA remains an orphan disease with no chemotherapeutic agents approved by the FDA for its treatment. AA has a unique biology in which it frequently forms diffuse peritoneal metastases, but almost never spreads via a hematogenous route and rarely spreads to lymphatics. Given its localization to the peritoneal space we hypothesized that intraperitoneal (IP) delivery of chemotherapy could be an effective treatment strategy. Here we tested the efficacy paclitaxel given by IP administration using three orthotopic PDX models of AA established in NSG mice. Weekly treatment of 25.0 mg/kg of IP paclitaxel dramatically reduced AA tumor growth in TM00351 (81.9% reduction vs. control), PMP-2 (98.3% reduction vs. control), and PMCA-3 (71.4% reduction vs. control) PDX models. Comparing the safety and efficacy of intravenous (IV) to IP administration in PMCA-3, neither 6.25 nor 12.5 mg/kg of IV paclitaxel significantly reduced tumor growth. These results suggest that IP administration of paclitaxel is favorable to IV administration. Given the established safety record of IP paclitaxel in gastric and ovarian cancers, and lack of effective chemotherapeutics for AA, these data showing the activity of IP paclitaxel in orthotopic PDX models of mucinous AA support the evaluation of IP paclitaxel in a prospective clinical trial.

Quantitative Dual-Energy CT Image Guidance for Thermochemical Ablation: In Vivo Results in the Rabbit VX2 Model.

PURPOSE: To evaluate the feasibility of using dual-energy computed tomography (CT) and theranostic cesium hydroxide (CsOH) for image guidance of thermochemical ablation (TCA) in a rabbit VX2 tumor model. MATERIALS AND METHODS: In vivo experiments were performed on New Zealand white rabbits, where VX2 tumor fragments (0.3 mL) were inoculated into the right and left flanks (n = 16 rabbits, 32 tumors). Catheters were placed in the approximate center of 1- to 2-cm diameter tumors under ultrasound guidance. TCA was delivered in 1 of 3 treatment groups: untreated control, 5-M TCA, or 10-M TCA. The TCA base reagent was doped with 250-mM CsOH. Dual-energy CT was performed before and after TCA. Cesium (CS)-specific images were postprocessed on the basis of previous phantom calibrations to determine Cs concentration. Line profiles were drawn through the ablation center. Twenty-four hours after TCA, subjects were euthanized, and the resulting damage was evaluated with histopathology. RESULTS: Cs was detected in 100% of treated tumors (n = 21). Line profiles indicated highest concentrations at the injection site and decreased concentrations at the tumor margins, with no Cs detected beyond the ablation zone. The maximum detected Cs concentration ranged from 14.39 to 137.33 mM. A dose-dependent trend in tissue necrosis was demonstrated between the 10-M TCA and 5-M TCA treatment groups (P = .0005) and untreated controls (P = .0089). CONCLUSIONS: Dual-energy CT provided image guidance for delivery, localization, and quantification of TCA in the rabbit VX2 model.

Bismuth Nanoparticle and Polyhydroxybutyrate Coatings Enhance the Radiopacity of Absorbable Inferior Vena Cava Filters for Fluoroscopy-Guided Placement and Longitudinal Computed Tomography Monitoring in Pigs.

Inferior vena cava filters (IVCFs) constructed with poly-p-dioxanone (PPDO) are promising alternatives to metallic filters and their associated risks and complications. Incorporating high-Z nanoparticles (NPs) improves PPDO IVCFs' radiopacity without adversely affecting their safety or performance. However, increased radiopacity from these studies are insufficient for filter visualization during fluoroscopy-guided PPDO IVCF deployment. This study focuses on the use of bismuth nanoparticles (BiNPs) as radiopacifiers to render sufficient signal intensity for the fluoroscopy-guided deployment and long-term CT monitoring of PPDO IVCFs. The use of polyhydroxybutyate (PHB) as an additional layer to increase the surface adsorption of NPs resulted in a 2-fold increase in BiNP coating (BiNP-PPDO IVCFs, 3.8%; BiNP-PPDO + PHB IVCFs, 6.2%), enabling complete filter visualization during fluoroscopy-guided IVCF deployment and, 1 week later, clot deployment. The biocompatibility, clot-trapping efficacy, and mechanical strength of the control PPDO (load-at-break, 6.23 ± 0.13 kg), BiNP-PPDO (6.10 ± 0.09 kg), and BiNP-PPDO + PHB (6.15 ± 0.13 kg) IVCFs did not differ significantly over a 12-week monitoring period in pigs. These results indicate that BiNP-PPDO + PHB can increase the radiodensity of a novel absorbable IVCF without compromising device strength. Visualizing the device under conventional radiographic imaging is key to allow safe and effective clinical translation of the device.

Alterations of the Mdm2 C-Terminus Differentially Impact Its Function In Vivo.

Murine double minute 2 (Mdm2) is the principal E3-ubiquitin ligase for p53 and contains a C2H2C4 type RING domain wherein the last cysteine residue is followed by an evolutionarily conserved 13 amino acid C-terminal tail. Previous studies have indicated that integrity of the C-terminal tail is critical for Mdm2 function. Recently, a mutation extending the MDM2 length by five amino acids was identified and associated with enhanced p53 response in fibroblasts and premature aging in a human patient. To investigate the importance of the conserved Mdm2 C-terminal length on p53 regulatory function in vivo, we engineered three novel mouse alleles using CRISPR-Cas9 technology. Genetic studies with these murine models showed that curtailing Mdm2 C-terminal length by even a single amino acid leads to p53-dependent embryonic lethality. Extension of the Mdm2 C-terminal length by five amino acids (QLTCL) yielded viable mice that are smaller in size, exhibit fertility problems, and have a shortened life span. Analysis of early passage mouse embryonic fibroblasts indicated impaired Mdm2 function correlates with enhanced p53 activity under stress conditions. Furthermore, analysis in mice showed tissue-specific alterations in p53 target gene expression and enhanced radiosensitivity. These results confirm the physiological importance of the evolutionarily conserved Mdm2 C-terminus in regulating p53 functions. SIGNIFICANCE: This in vivo study highlights that alterations to the C-terminus of Mdm2 perturb its regulation of the tumor suppressor p53.

Technical Note: Histological validation of anatomical imaging for breast modeling using a novel cryo-microtome.

PURPOSE: Precise correlation between three-dimensional (3D) imaging and histology can aid biomechanical modeling of the breast. We develop a framework to register ex vivo images to histology using a novel cryo-fluorescence tomography (CFT) device. METHODS: A formalin-fixed cadaveric breast specimen, including chest wall, was subjected to high-resolution magnetic resonance (MR) imaging. The specimen was then frozen and embedded in an optimal cutting temperature (OCT) compound. The OCT block was placed in a CFT device with an overhead camera and 50 µm thick slices were successively shaved off the block. After each shaving, the block-face was photographed. At select locations including connective/adipose tissue, muscle, skin, and fibroglandular tissue, 20 µm sections were transferred onto cryogenic tape for manual hematoxylin and eosin staining, histological assessment, and image capture. A 3D white-light image was automatically reconstructed from the photographs by aligning fiducial markers embedded in the OCT block. The 3D MR image, 3D white-light image, and photomicrographs were rigidly registered. Target registration errors (TREs) were computed based on 10 pairs of points marked at fibroglandular intersections. The overall MR-histology registration was used to compare the MR intensities at tissue extraction sites with a one-way analysis of variance. RESULTS: The MR image to CFT-captured white-light image registration achieved a mean TRE of 0.73 ± 0.25 mm (less than the 1 mm MR slice resolution). The block-face white-light image and block-face photomicrograph registration showed visually indistinguishable alignment of anatomical structures and tissue boundaries. The MR intensities at the four tissue sites identified from histology differed significantly (p < 0.01). Each tissue pair, except the skin-connective/adipose tissue pair, also had significantly different MR intensities (p < 0.01). CONCLUSIONS: Fine sectioning in a highly controlled imaging/sectioning environment enables accurate registration between the MR image and histology. Statistically significant differences in MR signal intensities between histological tissues are indicators for the specificity of correlation between MRI and histology.

The Provocative Roles of Platelets in Liver Disease and Cancer.

Both platelets and the liver play important roles in the processes of coagulation and innate immunity. Platelet responses at the site of an injury are rapid; their immediate activation and structural changes minimize the loss of blood. The majority of coagulation proteins are produced by the liver-a multifunctional organ that also plays a critical role in many processes: removal of toxins and metabolism of fats, proteins, carbohydrates, and drugs. Chronic inflammation, trauma, or other causes of irreversible damage to the liver can dysregulate these pathways leading to organ and systemic abnormalities. In some cases, platelet-to-lymphocyte ratios can also be a predictor of disease outcome. An example is cirrhosis, which increases the risk of bleeding and prothrombotic events followed by activation of platelets. Along with a triggered coagulation cascade, the platelets increase the risk of pro-thrombotic events and contribute to cancer progression and metastasis. This progression and the resulting tissue destruction is physiologically comparable to a persistent, chronic wound. Various cancers, including colorectal cancer, have been associated with increased thrombocytosis, platelet activation, platelet-storage granule release, and thrombosis; anti-platelet agents can reduce cancer risk and progression. However, in cancer patients with pre-existing liver disease who are undergoing chemotherapy, the risk of thrombotic events becomes challenging to manage due to their inherent risk for bleeding. Chemotherapy, also known to induce damage to the liver, further increases the frequency of thrombotic events. Depending on individual patient risks, these factors acting together can disrupt the fragile balance between pro- and anti-coagulant processes, heightening liver thrombogenesis, and possibly providing a niche for circulating tumor cells to adhere to-thus promoting both liver metastasis and cancer-cell survival following treatment (that is, with minimal residual disease in the liver).

Development and characterization of prototypes for in vitro and in vivo mouse models of ibrutinib-resistant CLL.

Although ibrutinib improves the overall survival of patients with chronic lymphocytic leukemia (CLL), some patients still develop resistance, most commonly through point mutations affecting cysteine residue 481 (C481) in Bruton's tyrosine kinase (BTKC481S and BTKC481R). To enhance our understanding of the biological impact of these mutations, we established cell lines that overexpress wild-type or mutant BTK in in vitro and in vivo models that mimic ibrutinib-sensitive and -resistant CLL. MEC-1 cell lines stably overexpressing wild-type or mutant BTK were generated. All cell lines coexpressed GFP, were CD19+ and CD23+, and overexpressed BTK. Overexpression of wild-type or mutant BTK resulted in increased signaling, as evidenced by the induction of p-BTK, p-PLCγ2, and p-extracellular signal-related kinase (ERK) levels, the latter further augmented upon IgM stimulation. In all cell lines, cell cycle profiles and levels of BTK expression were similar, but the RNA sequencing and reverse-phase protein array results revealed that the molecular transcript and protein profiles were distinct. To mimic aggressive CLL, we created xenograft mouse models by transplanting the generated cell lines into Rag2-/-γc-/- mice. Spleens, livers, bone marrow, and peripheral blood were collected. All mice developed CLL-like disease with systemic involvement (engraftment efficiency, 100%). We observed splenomegaly, accumulation of leukemic cells in the spleen and liver, and macroscopically evident necrosis. CD19+ cells accumulated in the spleen, bone marrow, and peripheral blood. The overall survival duration was slightly lower in mice expressing mutant BTK. Our cell lines and murine models mimicking ibrutinib-resistant CLL will serve as powerful tools to test reversible BTK inhibitors and novel, non-BTK-targeted therapeutics.

Meningoencephalomyelitis in domestic cats: 3 cases of Pasteurella multocida infection and literature review.

Neurologic diseases are common in domestic cats, and infectious agents are suspected to be the primary cause in 30-45% of cases. Among infectious etiologies, those of bacterial origin are only sporadically characterized in the literature, with few of these reports correlating gross and histologic findings with confirmatory bacteriologic identification. Here, we describe bacterial meningitis and meningoencephalomyelitis associated with Pasteurella multocida in 3 domestic cats. Purulent exudate expanding the cerebral meninges was grossly evident in 2 of the cases. In all 3 cases, histologic changes included multifocal suppurative-to-necrosuppurative meningitis and/or meningoencephalomyelitis of variable severity. Intralesional colonies of gram-negative, short rod-shaped to coccobacillary bacteria were evident histologically in only 1 case. P. multocida was confirmed by routine bacteriologic culture in all cases. Based on our cases, we hypothesize that the upper respiratory system serves as the main portal of entry for P. multocida, leading to invasion of the central nervous system and possible systemic hematogenous dissemination. A case series of meningoencephalomyelitis associated with P. multocida infection in cats has not been reported previously, to our knowledge. We also review briefly other causes of meningoencephalomyelitis in cats.

Nonphosphorylatable PEA15 mutant inhibits epithelial-mesenchymal transition in triple-negative breast cancer partly through the regulation of IL-8 expression.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that lacks targeted therapies. Patients with TNBC have a very poor prognosis because the disease often metastasizes. New treatment approaches addressing drivers of metastasis and tumor growth are crucial to improving patient outcomes. Developing targeted gene therapy is thus a high priority for TNBC patients. PEA15 (phosphoprotein enriched in astrocytes, 15 kDa) is known to bind to ERK, preventing ERK from being translocated to the nucleus and hence blocking its activity. The biological function of PEA15 is tightly regulated by its phosphorylation at Ser104 and Ser116. However, the function and impact of phosphorylation status of PEA15 in the regulation of TNBC metastasis and in epithelial-to-mesenchymal transition (EMT) are not well understood. METHODS: We established stable cell lines overexpressing nonphosphorylatable (PEA15-AA) and phospho-mimetic (PEA15-DD) mutants. To dissect specific cellular mechanisms regulated by PEA15 phosphorylation status, we performed RT-PCR immune and metastasis arrays. In vivo mouse models were used to determine the effects of PEA15 phosphorylation on tumor growth and metastasis. RESULTS: We found that the nonphosphorylatable mutant PEA15-AA prevented formation of mammospheres and expression of EMT markers in vitro and decreased tumor growth and lung metastasis in in vivo experiments when compared to control, PEA15-WT and phosphomimetic PEA15-DD. However, phosphomimetic mutant PEA15-DD promoted migration, mesenchymal marker expression, tumorigenesis, and lung metastasis in the mouse model. PEA15-AA-mediated inhibition of breast cancer cell migratory capacity and tumorigenesis was the partial result of decreased expression of interleukin-8 (IL-8). Further, we identified that expression of IL-8 was possibly mediated through one of the ERK downstream molecules, Ets-1. CONCLUSIONS: Our results show that PEA15 phosphorylation status serves as an important regulator for PEA15's dual role as an oncogene or tumor suppressor and support the potential of PEA15-AA as a therapeutic strategy for treatment of TNBC.

Targeting the αv integrin/TGF-ß axis improves natural killer cell function against glioblastoma stem cells.

Glioblastoma multiforme (GBM), the most aggressive brain cancer, recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. We showed that GSCs, but not normal astrocytes, are sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. Mass cytometry and single-cell RNA sequencing of primary tumor samples revealed that GBM tumor-infiltrating NK cells acquired an altered phenotype associated with impaired lytic function relative to matched peripheral blood NK cells from patients with GBM or healthy donors. We attributed this immune evasion tactic to direct cell-to-cell contact between GSCs and NK cells via αv integrin-mediated TGF-ß activation. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-ß signaling or with TGFBR2 gene-edited allogeneic NK cells prevented GSC-induced NK cell dysfunction and tumor growth. These findings reveal an important mechanism of NK cell immune evasion by GSCs and suggest the αv integrin/TGF-ß axis as a potentially useful therapeutic target in GBM.

Combining AFM13, a Bispecific CD30/CD16 Antibody, with Cytokine-Activated Blood and Cord Blood-Derived NK Cells Facilitates CAR-like Responses Against CD30+ Malignancies.

PURPOSE: Natural killer (NK)-cell recognition and function against NK-resistant cancers remain substantial barriers to the broad application of NK-cell immunotherapy. Potential solutions include bispecific engagers that target NK-cell activity via an NK-activating receptor when simultaneously targeting a tumor-specific antigen, as well as enhancing functionality using IL12/15/18 cytokine pre-activation. EXPERIMENTAL DESIGN: We assessed single-cell NK-cell responses stimulated by the tetravalent bispecific antibody AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on various types of NK cells using mass cytometry and cytotoxicity assays. The combination of AFM13 and IL12/15/18 pre-activation of blood and cord blood-derived NK cells was investigated in vitro and in vivo. RESULTS: We found heterogeneity within AFM13-directed conventional blood NK cell (cNK) responses, as well as consistent AFM13-directed polyfunctional activation of mature NK cells across donors. NK-cell source also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from patients with Hodgkin lymphoma. IL12/15/18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30+ lymphoma targets directed by AFM13, compared with cNK cells. Cord-blood NK cells preactivated with IL12/15/18 and ex vivo expanded with K562-based feeders also exhibited enhanced killing with AFM13 stimulation via upregulation of signaling pathways related to NK-cell effector function. AFM13-NK complex cells exhibited enhanced responses to CD30+ lymphomas in vitro and in vivo. CONCLUSIONS: We identify AFM13 as a promising combination with cytokine-activated adult blood or cord-blood NK cells to treat CD30+ hematologic malignancies, warranting clinical trials with these novel combinations.

LMW cyclin E and its novel catalytic partner CDK5 are therapeutic targets and prognostic biomarkers in salivary gland cancers.

Salivary gland cancers (SGCs) are rare yet aggressive malignancies with significant histological heterogeneity, which has made prediction of prognosis and development of targeted therapies challenging. In majority of patients, local recurrence and/or distant metastasis are common and systemic treatments have minimal impact on survival. Therefore, identification of novel targets for treatment that can also be used as predictors of recurrence for multiple histological subtypes of SGCs is an area of unmet need. In this study, we developed a novel transgenic mouse model of SGC, efficiently recapitulating the major histological subtype (adenocarcinomas of the parotid gland) of human SGC. CDK2 knock out (KO) mice crossed with MMTV-low molecular weight forms of cyclin E (LMW-E) mice generated the transgenic mouse models of SGC, which arise in the parotid region of the salivary gland, similar to the common site of origin seen in human SGCs. To identify the CDK2 independent catalytic partner(s) of LMW-E, we used LMW-E expressing cell lines in mass spectrometric analysis and subsequent biochemical validation in pull down assays. These studies revealed that in the absence of CDK2, LMW-E preferentially binds to CDK5. Molecular targeting of CDK5, using siRNA, resulted in inhibition of cell proliferation of human SGCs overexpressing LMW-E. We also provide clinical evidence of significant association of LMW-E/CDK5 co-expression and decreased recurrence free survival in human SGC. Immunohistochemical analysis of LMW-E and CDK5 in 424 patients representing each of the four major histological subtypes of human salivary cancers (Aci, AdCC, MEC, and SDC) revealed that LMW-E and CDK5 are concordantly (positive/positive or negative/negative) expressed in 70% of these patients. The co-expression of LMW-E/CDK5 (both positive) robustly predicts the likelihood of recurrence, regardless of the histological classification of these tumors. Collectively, our results suggest that CDK5 is a novel and targetable biomarker for the treatment of patients with SGC presenting with LMW-E overexpressing tumors.

Dual Inhibition of MEK and AXL Targets Tumor Cell Heterogeneity and Prevents Resistant Outgrowth Mediated by the Epithelial-to-Mesenchymal Transition in NSCLC.

The epithelial-to-mesenchymal transition (EMT) is a dynamic epigenetic reprogramming event that occurs in a subset of tumor cells and is an initiating step toward invasion and distant metastasis. The process is reversible and gives plasticity to cancer cells to survive under variable conditions, with the acquisition of cancer stem cell-like characteristics and features such as drug resistance. Therefore, understanding survival dependencies of cells along the phenotypic spectrum of EMT will provide better strategies to target the spatial and temporal heterogeneity of tumors and prevent their ability to bypass single-inhibitor treatment strategies. To address this, we integrated the data from a selective drug screen in epithelial and mesenchymal KRAS/p53 (KP)-mutant lung tumor cells with separate datasets including reverse-phase protein array and an in vivo shRNA dropout screen. These orthogonal approaches identified AXL and MEK as potential mesenchymal and epithelial cell survival dependencies, respectively. To capture the dynamicity of EMT, incorporation of a dual fluorescence EMT sensor system into murine KP lung cancer models enabled real-time analysis of the epigenetic state of tumor cells and assessment of the efficacy of single agent or combination treatment with AXL and MEK inhibitors. Both two- and three-dimensional culture systems and in vivo models revealed that this combination treatment strategy of MEK plus AXL inhibition synergistically killed lung cancer cells by specifically targeting each phenotypic subpopulation. In conclusion, these results indicate that cotargeting the specific vulnerabilities of EMT subpopulations can prevent EMT-mediated drug resistance, effectively controlling tumor cell growth and metastasis. SIGNIFICANCE: This study shows that a novel combination of MEK and AXL inhibitors effectively bypasses EMT-mediated drug resistance in KRAS/p53-mutant non-small cell lung cancer by targeting EMT subpopulations, thereby preventing tumor cell survival.

Targeting a cytokine checkpoint enhances the fitness of armored cord blood CAR-NK cells.

Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2-containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation "armored" chimeric antigen receptor (CAR) engineering of cord blood-derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell antitumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15-secreting armored CAR-NK cells by promoting their metabolic fitness and antitumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.

Large-scale GMP-compliant CRISPR-Cas9-mediated deletion of the glucocorticoid receptor in multivirus-specific T cells.

Virus-specific T cells have proven highly effective for the treatment of severe and drug-refractory infections after hematopoietic stem cell transplant (HSCT). However, the efficacy of these cells is hindered by the use of glucocorticoids, often given to patients for the management of complications such as graft-versus-host disease. To address this limitation, we have developed a novel strategy for the rapid generation of good manufacturing practice (GMP)-grade glucocorticoid-resistant multivirus-specific T cells (VSTs) using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology. We have shown that deleting the nuclear receptor subfamily 3 group C member 1 (NR3C1; the gene encoding for the glucocorticoid receptor) renders VSTs resistant to the lymphocytotoxic effect of glucocorticoids. NR3C1-knockout (KO) VSTs kill their targets and proliferate successfully in the presence of high doses of dexamethasone both in vitro and in vivo. Moreover, we developed a protocol for the rapid generation of GMP-grade NR3C1 KO VSTs with high on-target activity and minimal off-target editing. These genetically engineered VSTs promise to be a novel approach for the treatment of patients with life-threatening viral infections post-HSCT on glucocorticoid therapy.